Reassessment of the microbicidal activity of reactive oxygen species and hypochlorous acid with reference to the phagocytic vacuole of the neutrophil granulocyte.

Centre for Molecular Medicine, University College London, 5 University Street, London WC1E 6JJ, UK.

Abstract

During phagocytosis, neutrophils undergo a burst of respiration in which oxygen is reduced to superoxide (O(-)(2)), which dismutates to form H(2)O(2). Myeloperoxidase (MPO) is discharged from the cytoplasmic granules into the phagosome following particle ingestion. It is thought to utilize H(2)O(2) to oxidize halides, which then react with and kill ingested microbes. Recent studies have provided new information as to the concentration of O(-)(2) and proteins, and the pH, within the vacuole. This study was conducted to examine the antimicrobial effect of O(-)(2), H(2)O(2) and hypochlorous acid under these conditions and it was found that the previously described bactericidal effect of these agents was reversed in the presence of granule proteins or MPO. To establish which cellular proteins were iodinated by MPO, cellular proteins and bacterial proteins, iodinated in neutrophils phagocytosing bacteria in the presence of (125)I, were separated by 2D gel electrophoresis. Iodinated spots were detected by autoradiography and the oxidized proteins were identified by MS. The targets of these iodination reactions were largely those of the host cell rather than those of the engulfed microbe.

Effect of pH on bacterial viability, destruction by stimulated neutrophils and bactericidal effects of . (a) IgG opsonized S. aureus (■) or E. coli (□) (1 × 108 c.f.u. ml−1) was mixed at a ratio of one target organism to five neutrophils in 1 ml PBS (pH 7·3) for the indicated periods of time and bacterial viability was determined. The mean (±se) of three experiments is shown. No significant difference was observed between killing of S. aureus and E. coli. (b) Bactericidal effect of was determined by suspending S. aureus (1 × 107 c.f.u.) in 0· 01 M phosphate buffer (pH 7·5) (●) or buffer containing different concentrations of KO2 (○). Each point is the mean of triplicate experiments (±se). (c, d) To determine the effect of pH on bacterial viability, S. aureus or E. coli (1 × 107 c.f.u. ml−1) was incubated at 37 °C in 0·01 M phosphate buffer, pH 5·5 (□), 6·5 (○) or 7·5 (●). Reduction in survival of S. aureus at pH 5·5 compared to 6·5 was found to be significant, P < 0·033.